In previous work, we have used injection of plasmid DNA into the nuclei of mouse oocytes, fertilized eggs (1-cell embryos) and 2-cell embryos in an effort to identify cis-acting sequences and trans-acting factors that are required to activate DNA replication and gene expression at the beginning of mammalian development. DNA replication first occurs in 1-cell mouse embryos, and zygotic gene expression in 2-cell mouse embryos. These results, together with studies on endogenous gene expression and nuclear transplantation, revealed several features that regulate DNA replication and gene expression at the beginning of mammalian development, identified specific regulatory sequences and factors, and demonstrated the developmental acquisition of specific regulatory functions [Nothias et al. J. Biol. Chem. 270: 22077-80 (1995); Majumder & DePamphilis, BioEssays 17: 879-89 (1995)]. A time dependent mechanism ("zygotic clock") delays transcription of zygotic genes and translation of nascent mRNA until the 2-cell stage in development. This allows chromatin remodeling to occur on the paternal and maternal genomes without accidentally expressing genes prematurely. Just prior to activation of zygotic genes in 2-cell embryos, changes in chromatin structure occur that mediate a general repression of promoter and origin activities. These changes involve changes in the pattern of histone synthesis and acetylation. This repression can be relieved by linking promoters or replication origins to embryo responsive enhancers. However, the ability to use these enhancers does not appear until formation of 2-cell embryo, because an enhancer specific co-activator is absent prior to this stage in development. Therefore, the first two S-phases in mouse development occur under conditions where enhancers cannot function, a fact that may contribute to developmental acquisition of site specific initiation of DNA replication at later stages in development. Another developmentally acquired regulatory element is the TATA-box. The need for a TATA-box to facilitate promoter activity appears only after cell differentiation occurs and then only as a mediator of enhancer function. Other transcription factor binding sites, such as Sp1, are utilized throughout development. Specific DNA sequences that bind TEAD transcription factors provide a powerful enhancer activity in cleavage stage embryos. Mice encode four TEAD genes with the same DNA binding domain and affinity for DNA. All four mTEAD genes are expressed at later embryonic stages, and virtually every tissue expresses at least one family member, but only mTEAD-2 is expressed during the first 7 days of embryonic development. Thus, mTEAD-2 is one of the first transcription factors known to be expressed during zygotic gene activation, presumably turning on the next cascade of genes in the developmental program. Studies are now in progress to identify the role of TEAD-2 in mammalian development, and to identify the regulatory sequences that permit TEAD-2 to be expressed at the onset of ZGA while delaying the expression of TEAD-1, 3 and 4 to much later in development.